Methods for treating parasitic infection using thiopeptides

ABSTRACT

The present invention provides a method for treating a parasitic infection in a subject infected with a parasite having a plastid-like organelle, comprising administering to the subject an infection treating amount of a thiopeptide in a pharmaceutically acceptable carrier. Methods for treating Cryptospordium, Toxoplasma or Plasmodium infection in a subject are also provided, each method comprising administering to the subject an infection treating amount of a thiopeptide in a pharmaceutically acceptable carrier.

This application claims priority to and is a national phase applicationfiled under 35 U.S.C. § 371 of International Application No.PCT/US97/11939, filed Jul. 7, 1997, which application is incorporatedherein in its entirety by reference.

This invention was made with government support under the NationalInstitutes of Health Intramural Research Program. The government hascertain rights in this invention.

BACKGROUND ART

1. Field of the Invention

The present invention provides methods for treating parasitic infectionsin animals using thiopeptides. In particular, this invention relates tothe treatment of parasitic infections caused by members of the phylumApicomplexa (e.g., Cryptospordium, Plasmodium, Toxoplasma) byadministering thiopeptides in a pharmaceutically acceptable carrier.

2. Background Art

Thiopeptides are sulfur-rich peptide antibiotics containing multiplethiazole rings which are naturally produced by streptomycetes (37).These antibiotics, of which thiostrepton is an example, inhibittranslation and ribosomal GTPase activity by binding to a limited andconserved region in the large subunit (LSU) rRNA found in eubacteria andorganelles and not the corresponding region in eucarya (12-14).

Plasmodium, the agent responsible for malaria, is an obligateintracellular parasite. More than ten years ago an urgent need for drugsagainst malaria was identified (33). The antibiotics currently in use,including the tetracyclines and clindamycin, for the treatment andprophylaxis of malaria have little action on pre-erythrocytic stages anda slow action on blood stages, but are used for treatment of drugresistant strains because of their safety rather than their efficacy(34,35). Furthermore, the rapid spread of resistance to chloroquine hasheightened the need for ready availability of relatively low costprophylactic and therapeutic anti-malarial drugs. These includecompounds that reverse resistance to chloroquine, compounds that actrapidly to treat falciparum malaria and others that can be administeredby methods other than injection (to avoid the use of contaminatedneedles).

Human clinical cryptosporidiosis infection varies with host immunecompetence from mild, self-limiting diarrhea to life-threateningenteritis complicated by extraintestinal disease. There is no reliabletherapy for cryptosporidiosis. The problems of developing in vitro andin vivo methods of screening drugs, such as limited availability andpoor reproducibility, have contributed to this lack of effectivetreatment. However, the major hindrance has been a lack of understandingof the parasite, its virulence and its interactions with the host'simmune system (42).

The present invention overcomes previous shortcomings in developingeffective treatments of these types of parasitic infections by providinga method for treating such infections in subjects caused by parasiteshaving a plastid-like organelle by administering thiopeptides to thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amount of oocysts shed after administering thiostrepton(500 mg/kg/day) or placebo to scid mice preconditioned with monoclonalantibodies against interferon-γ and orally infected with 10⁷Cryptospordium parvum oocysts.

SUMMARY OF THE INVENTION

The present invention provides a method for treating a parasiticinfection in a subject infected with a parasite having a plastid-likeorganelle, comprising administering to the subject an infection treatingamount of a thiopeptide in a pharmaceutically acceptable carrier.

Methods are also provided for treating Cryptospordium, Toxoplasma orPlasmodium infection in a subject, each method comprising administeringto the subject an infection treating amount of a thiopeptide in apharmaceutically acceptable carrier.

Various other objectives and advantages of the present invention willbecome apparent from the following description.

DETAILED DESCRIPTION OF THE INVENTION

As used in the claims, “a” can include one or more.

The present invention provides a method for treating a parasiticinfection in a subject infected with a parasite having a plastid-likeorganelle, comprising administering to the subject an infection treatingamount of a thiopeptide in a pharmaceutically acceptable carrier. Thesubject of the invention can be any animal which can become infected bya parasite having a plastid-like organelle. For example, the animal ofthis invention can be, but is not limited to, cows, sheep, goats, pigs,birds (e.g., ducks, geese, turkeys, chickens) and shellfish. In apreferred embodiment, the animal can be a mammal and most preferably isa human. As used herein, the term “plastid-like organelle” means acircular, extrachromosomal DNA of eubacterial origin residing in amembrane-bound organelle, sharing features such as ribosomal RNA (rRNA)and transfer RNA (tRNA) gene organization with the plastids found inEuglena, red algae and green algae. The plastid-like organelle is alsoknown as an apiplast (Apicomplexan plastid).

The parasite of the present invention can be any parasite now known orlater identified to have the plastid-like organelle of the presentinvention. For example, the parasite of the invention can be of, but isnot limited to, the Apicomplexa phylum such as, for example, Babesia,Toxoplasma, Plasmodium, Eimeria, Isospora, Atoxoplasma, Cystoisospora,Hammondia, Besniotia, Sarcocystis, Frenkelia, Haemoproteus,Leucocytozoon, Theileria, Perkinsus and Gregarina spp.; Pneumocystiscarinii; members of the Microspora phylum such as, for example, Nosema,Enterocytozoon, Encephalitozoon, Septata, Mrazelia, Amblyospora, Ameson,Glugea, Pleistophora and Microporidium spp.; and members of theAscetospora phylum such as, for example, Haplosporidium spp. (39), aswell as any other parasite identified as having a plastid-like organelleof the present invention

The thiopeptide of the invention can be any member of the class ofcompounds characterized as sulfur-rich peptide antibiotics with multiplethiazole rings (37) now known or later identified to inhibit proteinsynthesis in the plastid-like organelle of parasites. For example, thethiopeptide can be, but is not limited to, thiostrepton:Ile-Ala-Ser-Ala-Ser-Cys-Thr-Thr-DCys-Ile-Cys-Thr-Cys-Ser-Cys-Ser-Ser-Ser(SEQ ID NO:8) (also known as A-8506, antibiotic 6761-31, antibiotic A8506, antibiotic X 146, bryamycin, thiactin and X 146), micrococcin P,hosiheptide (also known as multhiomycin), siomycin, sporangiomycin,althiomycin, the thiociffins and/or thiopeptin, as well as any othersulfur-rich peptide antibiotic containing multiple thiazolerings,produced by streptomycetes or other peptide antibiotic-producingorganisms.

To treat a parasitic infection caused by a parasite having aplastid-like organelle, the thiopeptide of the present invention can beadministered to the subject orally, parenterally, intranasally (i.e., byaerosol) or topically. The thiopeptide can be in a pharmaceuticallyacceptable carrier. By “pharmaceutically acceptable” is meant a materialthat is not biologically or otherwise undesirable, i.e., the materialmay be administered to a subject, along with the thiopeptide withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to mninimize any adverse side effects in the subject.

To treat a parasitic blood-stage infection (e.g., a malaria or babesiainfection), parenteral administration can be the preferred mode.Suitable carriers for parenteral administration of the thiopeptide in asterile solution or suspension can include sterile saline that maycontain additives, such as ethyl oleate or isopropyl myristate, and canbe injected, for example, intravenously, as well as into subcutaneous orintramuscular tissues.

Suitable carriers for oral administration include one or more substanceswhich may also act as flavoring agents, lubricants, suspending agents,or as protectants. Suitable solid carriers include calcium phosphate,calcium carbonate, magnesium stearate, sugars, starch, gelatin,cellulose, carboxypolymethylene, or cyclodextrans. Suitable liquidcarriers may be water, pyrogen free saline, pharmaceutically acceptedoils, or a mixture of any of these. The liquid can also contain othersuitable pharmaceutical additions such as buffers, preservatives,flavoring agents, viscosity or osmo-regulators, stabilizers orsuspending agents. Examples of suitable liquid carriers include waterwith or without various additives, including carboxypolymethylene as apH-regulated gel.

The thiopeptides of the present invention can be administered to thesubject in amounts sufficient to treat the parasitic infection in thesubject as desired. Optimal dosages used will vary according to theindividual and the particular parasitic infection, on the basis of age,size, weight, condition, etc, as well as the particular treatment effectbeing induced. One skilled in the art will realize that dosages are bestoptimized by the practicing physician and methods for determining dosageare described, for example, in Remington's Pharmaceutical Sciences (36).

In a preferred embodiment, the thiopeptide of the present invention canbe administered to a human or a non-human animal in a pharmaceuticallyacceptable carrier in a dosage range of about 50 to 550 mg/kg/day and ispreferably administered in a dosage of about 500 mg/kg/day. Treatmentcan be continued for an indefinite period of time, as indicated bymonitoring of the signs, symptoms and clinical parameters associatedwith the parasitic infection according to protocols standard in the artfor monitoring parasitic infections. Examples of the parameters thatwould be monitored can include, but are not limited to, amount andfrequency of diarrheal excretion, oocyst excretion, culture of theparasite in body fluids and tissues, body weight and blood chemistry andurine analysis of hepatobiliary function. Oocyst excretion can bemeasured by quantitation of acid-fast stained stool specimens, ELISAantigen capture, immunofluorescence assay, DNA amplification, etc.,according to protocols well known in the art.

In a particular embodiment, the present invention provides a method fortreating Cryptospordium infection in a subject, preferably human,comprising administering to the subject an infection treating amount ofa thiopeptide in a pharmaceutically acceptable carrier. To treat aCryptoporidium infection, the thiopeptide of this invention wouldpreferably be administered to the subject orally.

In another embodiment, the present invention provides a method fortreating Plasmodium infection in a subject, preferably human, comprisingadministering to the subject an infection treating amount of athiopeptide in a pharmaceutically acceptable carrier. To treat aPlasmodium infection, the thiopeptide of this invention would preferablybe administered to the subject parenterally.

A further embodiment of the present invention provides a method fortreating Toxoplasma infection in a subject, preferably human, comprisingadministering to the subject an infection treating amount of athiopeptide in a pharmaceutically acceptable carrier. To treat aToxoplasma infection, the thiopeptide of this invention would preferablybe administered to the subject parenterally.

It is also contemplated that the thiopeptide of the present inventioncan be administered in combination with other thiopeptides and/or otherantibiotics, in particular, peptidyl transferase inhibitors (40) such asamicetin, anisomycin and chloraamphenicol to treat a parasitic infectionin a subject. Thus, the present invention provides a compositioncomprising a thiopeptide and a peptidyl transferase inhibitor in apharmaceutically acceptable carrier, such as a composition comprisingthiostrepton and amicetin, thiostrepton and anisomycin, thiostrepton andchloramphenicol and the like.

Other antibiotics which can be combined with the thiopeptides of theclaimed invention can include, but are not limited to, paromomycin,azithromycin, clarithromycin, nitazoxanide, novobiocin, fusidic acid,nalidixic acid, doxycycline, immune globulin preparations and severalmalarial compounds (e.g., mefloquine and halofantine and their analogs,pentanidine and its analogs) for treating Cryptospordium infection;pyrimethamine, sulfadiazine, atovaquine, fusidic acid and rifbutin fortreating Toxoplasma infection; bactrim (trimethoprim/sulfa), atovaquoneand pentamidine for treating Pneumocystis infection; and monensin,salinomycin, diclazuril, lasalocid, robenidine, nicarbazin, sinefunginand various ionophores for treating Eimeria infection. Thus, the presentinvention contemplates a composition comprising a thiopeptide and one ormore antibiotics identified to be effective in treating parasiticinfections, in a pharmaceutically acceptable carrier. This combinationcan be administered orally, parenterally, intranasally or topically asdescribed above for thiopeptide administration and the same parametersregarding treatment and dosage as described above can be applied.Compositions comprising the above novel combinations are provided.

Also contemplated for the present invention is a composition comprisinga thiopeptide (with or without other antibiotics) and an adjuvant toenhance the therapeutic or prophylactic effect of the thiopeptide. Theadjuvant can be selected by standard criteria based on the particularthiopeptide used, the mode of administration and the subject (45). Forexample, the composition can include Freund's complete adjuvant,Freund's incomplete adjuvant, aluminum hydroxide or any other adjuvantknown to enhance the therapeutic or prophylactic effect of thethiopeptide.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations wherein will be apparent to those skilledin the art.

EXAMPLES I. Studies on Plasmodium

Assay of Growth inhibition. Thiostrepton (1525 u/mg; Calbiochem) andanisomycin (Sigma) were dissolved at 100 mM in DMSO (Pierce). P.falciparum (strain 3D7) (44) was maintained in culture with humanerythrocytes (5% hematocrit) in RPMI-1640 (Life Technologies)supplemented with HEPES and sodium bicarbonate and human sera (10%)under standard conditions (18,19). The growth inhibition assay wasconducted as described (20). Briefly, the parasitemia was adjusted to0.1% parasitemia, 2.5% hematocrit and 200 μl aliquots placed in wells ofa microtitre dish. Serial dilutions of drugs were made in RPMI.Thiostrepton was diluted to 10 mM in DMSO before the serial dilutions inRPMI. Aliquots (20 μl) were added in triplicate to the cultures in themicrotitre plate, mixing well. At the highest concentrations (final0.2mM), thiostrepton precipitates. After incubation for 48 hours understandard conditions, [2,8-³H]-hypoxanthine Moravek Biochemicals, 12.5Ci/mmol) in RPMI (20 μl, 0.05 mCi/ml) was added to each well. Afterincubation for a further 24 hours, the cultures were lysed andincorporated radioactivity was measured with an automated counter.

Assay of Inhibition of Protein Synthesis. Assay for inhibition ofprotein synthesis was similar to above except the culture and drugdilutions were in RPMI-1640 without leucine (Select-Amine, LifeTechnologies) and the sera was extensively dialyzed as described (21).Also, the cultures were adjusted to 1% parasitemia prior to theexperiment. Microtitre plates were set up with drug dilutions asdescribed above except cultures were incubated with drug for only 4hours. [3,4,5-³H]-Leucine (Moravek Biochemicals, 122 Ci/mmol, 0.5mCi/ml, 100 μCi) was diluted to 4 ml in RPMI without leucine. Theradiolabeled leucine (20 μl, 1 mCi) was added to each well and theplates were further incubated for four hours. The microtitre plates wereprocessed as above to quantitate the incorporated radiolabel.

As of organelle and cyloplasmic transcript levels. RNA polymerasetranscript levels were assessed by comparing the amounts of RNAsynthesized at timepoints following drug treatment. Cultures of P.falciparum (3.2% parasitemia, 5% hematocrit) were treated withthiostrepton at 8 μM and rifampin (Sigma) at 80 μM; near the IC₉₉ values(this study and ref. 22). Aliquots (5 ml) of treated and controlcultures were removed and immediately processed for RNA with aguanidinium thiocyanate solution, according to the manufacturer'sdirections (RNAgents, Promega). All RNA samples were dissolved in 50 μlDEPC-treated water, DNase I treated as previously described (23) and analiquot (1 μl) was removed for RT-PCR First strand synthesis of cDNA wascompleted with a random hexamer (Superscript Preamplification System,Life Technologies). One-tenth of the CDNA product was utilized for PCRof rpoB/C, MSA1, and rRNA. Primers corresponding to the 3′ region ofrpoB, 5′-GGGCTTTAGAAGCTTTTGG-3′ (SEQ ID NO:1), and the 5′ region ofrpoC, 5′-CCATTTAAAATTGGTAATCCTG-3′ (SEQ ID NO:2) were applied asdescribed (2,3) for PCR of nascent rpoB/C transcripts. Reactions werecycled with the following parameters: 94° C./30 seconds, 42° C./30seconds, 72° C./60 seconds, 35 cycles. Primers for amplification ofnucleotide 64 to 614 of MSA1 with 5′-GTGTGATAATATTCATGG-3′ (SEQ ID NO:3)and 5′-GGAGAGCATTTGGTG-3′ (SEQ ID NO:4)(24) and the small subunit rRNAwith oligonucleotides 841 and 844 (23) were used for amplificationreactions following the parameters in the respective references exceptwith 35 cycles. Samples were also analyzed after 25 cycles ofamplification to ensure detection in the linear range of theamplification reaction, with similar results. Following electrophoresisof aliquots from the amplification reactions on 1% agarose:TBE gels,samples were transferred to nylon membranes (GeneScreen Plus, DuPont)and hybridized as described (25). The amplification products were probedwith 5′-³²P-labeled oligonucleotides. The rpoB/C products were probedwith 5′-GTTTAGCTATTAATATAGAAGC-3′ (SEQ ID NO:5) (nucleotide 2009-2030 ofrpoB) and 5′-CGGAGAGGTATTAATACC-3′ (SEQ ED NO:6) (nucleotide 108-125 ofrpoC), in 5×SSC, 10 mM sodium phosphate, 0.05% sodium pyrophosphate, 1%sodium dodecyl sulfate, 5×Denhardt's solution, 100 μg/ml yeast tRNA, 42°C. and washed in the hybridization solution lacking Denhardt's and tRNAat 37° C., three times. The final wash was 1×SSC, 0.5% sodium dodecylsulfate, 42° C. followed by autoradiography. The same results wereobtained with either probe. The MSA1 amplification products weresimilarly probed with 5′-AAACTTGTGTTCGGATATAG-3′ (SEQ ID NO:7) and therRNA products with oligonucleotide 842 (23).

Assay of inhibition. The effect of thiostrepton on growth and proteinsynthesis of P. falciparum was compared to anisomycin, since the effectof both of these drugs is on protein synthesis. To assay the effect ofthiostrepton and anisomycin on the growth of P. faciparum, inhibition ofthe uptake and incorporation of [³H]hypoxanthine was quantitated atserial dilutions of the drugs on an in vitro culture of P. falciparum.Both compounds inhibited growth in the micromolar range (IC₅₀, 1.8 μMfor thiostrepton and 0.5 μM for anisomycin). These values are comparableto previously published data for anisomycin (28). Inhibition of theincorporation of [³H]leucine was also tested at the same dilutions ofthe drugs. For anisomycin, total protein synthesis was inhibited 50% atthe IC₅₀ (0.511 μM) for inhibition of growth, with theconcentration-response almost superimposable on that of proteinsynthesis. However, for thiostrepton, only a negligible amount ofinhibition of total protein synthesis was observed at the IC₅₀ forgrowth. More than ten-fold higher concentrations than the IC₅₀ ofthiostrepton were required for almost complete inhibition of totalprotein synthesis. The lack of inhibition of protein synthesis withthiostrepton at the IC₅₀ for inhibition of growth suggested that theprincipal target for the drug is different than cytoplasmic proteinsynthesis. This would occur if organelle protein synthesis was thetarget of inhibition (29,30).

The target of inhibition by thiostrepton. In the absence of a directmeasure of plastid-like organelle protein synthesis, assay of mRNAlevels by RT/PCR provides a sensitive assay for the selective effect ofthiostrepton on the plastid-like organelle. The presence of a proteinencoded by the organelle, identified in the 35-kb genome as a homolog ofeubacterial RNA polymerase encoded by the rpoB and rpoc genes (5), wasassayed during treatment. Selective inhibition of the plastid-like RNApolymerase with rifampin provides a comparison with the effect ofthiostrepton, since prokaryotic RNA polymerases are sensitive torifampin. The synthesis of the rpoB/C mRNA encoded by the 35-kb genomewas then compared to a nuclear-encoded mRNA On the basis that the 35-kbencoded rpoB and rpoC are transcribed as a polycistronic mRNA (3),nascent transcripts were assayed at various timepoints during drugtreatment by RT/PCR of the mRNA including the intergenic spacer betweenrpoB/C. Also, the sensitivity of RT/PCR provides a relative estimate ofthe mRNAs corresponding to those encoded on the 35-kb genome versusthose of nuclear-encoded mRNAs. Amplification of part of the mRNAcorresponding with Merozoite Surface Antigen (MSA1) was chosen as anuclear-encoded mRNA, as this is abundant in erythrocytic stages of P.falciparum (24). As a control, a section of nuclear-encoded SSU rRNA wasalso amplified as this is unaffected by antibiotics.

The results show that thiostrepton and rifampin have similar effects onthe decay of mRNA corresponding to rpoB/C; occurring within 6 hours ofdrug treatment. A time course with thiostrepton showed a decline of therpoB/C product with time, with a notable affect after only one hour oftreatment. Within the duration of the experiment (8 hours), the level ofrpoB/C in untreated controls was consistent. Although thiostrepton maybe inhibiting a specific subset of nuclear-encoded mRNAs, as the plastidRNA polymerase is composed of subunits of both nuclear and organelleorigin, there was no effect on the levels of nuclear-encoded MSA mRNAnor total rRNA Since completion of the erythrocytic cycle for P.falciparum takes about 48 hours, it would expected that the effect onnuclear-encoded mRNAs would be observed only with longer time points andwould reflect cell death rather than specific targeting the plastid-likeorganelle. These data also indicated that rifampin is a specificinhibitor of the RNA polymerase encoded by the plastid-like organelle(31,32). The data presented here demonstrate that the target forthiostrepton is the LSU encoded by the 35-kb organelle, while the targetfor anisomycin is the nuclear-encoded LSU rRNA and perhapsorganelle-encoded LSU rRNAs. Although the function of the 35-kbplastid-like organelle is not known (5), inhibition of growth bythiostrepton indicates that protein synthesis from this organelle isessential for growth of the blood-stages of the parasite.

II. Studies on Cryposporidium

A. Experiment 1.

In vitro doses of drugs. Thiostrepton (Calbiochem) was dissolved insterile complete Dulbecco's Modified Eagle Medium (DMEM) supplementedwith dimethyl sulfoxide (DMSO) at 0.2% and tested at concentrations of800, 400, 200, and 100, 10, 1 and 0.1 μg/ml.

Toxicity Testing Assay. 200 μl of medium containing drug at theabove-mentioned concentrations and positive control preparations[paromomycin/DMSO (2 mg/ml/0.2%)] were introduced into two wells of a 96well plate containing confluent MDBKF5D2 cell monolayers (ATCC accessionnumber CCL-22) infected with intact C. parvum GCH1 oocysts (5.0×10⁴ perwell) (43) and two wells without monolayers. The drug was incubated onthe monolayers at 37° C. and 8% CO₂. At 48 hours, MTS (Owen's solution)and PMS were added to each well at concentrations of 333 μg/ml and 25 μMrespectively. The plate was returned to the incubator in the dark todevelop for two hours. At two hours, 100 μl of each supernatant wastransferred to a new microtiter plate and the optical density (OD) wasread in an ELISA plate reader at 490 nm.

Percent toxicity was calculated by subtracting the mean drug OD from themedium OD, divided by the medium OD, all of which was then multiplied by100, as shown in the equation below. Cytotoxicity scores were assignedas follows: 0-5% toxicity=0, 6-25% toxicity=1, 26-50% toxicity=2, 51-75%toxicity=3, and 76-100% toxicity=4. As a standard, cytotoxicity scoresof 0 or, 1 were considered acceptable levels of toxicity. Toxicityscores of 2, 3, or 4 were considered as high levels of toxicity to thecell monolayer.$\frac{{O\quad D\quad {medium}} - {O\quad D\quad {drug}}}{O\quad D\quad {medium}} \times 100$

Intact C. parvum oocyst asset 3.0×10⁴ C. parvum GCH1 oocysts per wellwere incubated in the above-mentioned concentrations of drug at 37° C.(8% CO₂) on confluent MDBKF5D2 cell monolayers in 96 well microtiterplates. The level of infection in each well was determined and analyzedby immunofluorescence assay at 48 hours, using C. parvum sporozoiterabbit anti-serum (0.1%) and fluorescein-conjugated goat anti-rabbitantibody (1.0%). Percent inhibition was calculated by subtracting themean parasite/drug from the mean parasitelmedium, divided by the meanparasite/medium, all of which was multiplied by 100. The analysis wasperformed using MCID and an inverted microscope.

Tables 1 and 2 represent the results of two separate experiments. Mediumand oocyst lysate toxicity levels are included. Thiostrepton appears toshow good activity at concentrations of 200 and above with littletoxicity to cells.

Experiment 2

In vitro doses of drugs. Thiostrepton (Calbiochem) and Phavic (NTZ) weredissolved in sterile complete Dulbecco's Modified Eagle Medium (DMEM)supplemented with dimethyl sulfoxide (DMSO) at 0.2% and tested atconcentrations of 800, 400, 200, and 100 μg/ml. NTZ was tested atconcentrations of 100, 10, 1 and 0.1 μg/ml. Paromomycin (Sigma) wasdissolved in DMEM and tested at concentrations of 2000, 1000, 500 and250 μg/ml.

Toxicity Testing Assay. 200 μl of medium containing drugs at theabove-mentioned concentrations and positive control preparations[paromomycin/DMSO (2 mg/ml/0.2%)] were introduced into two wells of a 96well plate containing confluent MDBKF5D2 cell monolayers infected withintact C. parvum GCH1 oocysts (5.0×10⁴ per well) and two wells withoutmonolayers. The drug was incubated on the monolayers at 37° C. and 8%CO₂. At 48 hours, MTS (Owen's solution) and PMS were added to each wellat concentrations of 333 μg/ml and 25μM respectively. The plate wasreturned to the incubator in the dark to develop for two hours. At twohours, 100 μl of each supernatant was transferred to a new microtiterplate and the optical density (OD) was read in an ELISA plate reader at490 nm.

Percent toxicity was calculated by subtracting the mean drug OD from themedium OD, divided by the medium OD, all of which was then multiplied by100, as shown in the equation below. Cytotoxicity scores were assignedas follows: 0-5% toxicity=0, 6-25% toxicity=1, 26-50% toxicity=2, 51-75%toxicity=3, and 76-100% toxicity=4. As a standard, cytotoxicity scoresof 0 or 1 were considered acceptable levels of toxicity. Toxicity scoresof 2, 3, or 4 were considered as high levels of toxicity to the cellmonolayer.$\frac{{O\quad D\quad {medium}} - {O\quad D\quad {drug}}}{O\quad D\quad {medium}} \times 100$

Intact C. parvum oocyst assay . 3.0×10⁴ C. parvum GCH1 oocysts per wellwere incubated in the above-mentioned concentrations of drug at 37° C.(8% CO₂) on confluent MDBKF5D2 cell monolayers in 96 well microtiterplates. For some monolayers, the oocysts were incubated in DMEM on thecell monolayers for four hours, at which time the monolayers were washedand then drug was added to the wells. The level of infection in eachwell was determined and analyzed by immunofluorescence assay at 48hours, using C. parvum sporozoite rabbit anti-serum (0.1%) andfluorescein-conjugated goat anti-rabbit antibody (1.0%). Percentinhibition was calculated by subtracting the mean parasite/drug from themean parasite/medium, divided by the mean parasite/medium, all of whichwas multiplied by 100. The analysis was performed using MCID and aninverted microscope.

Tables 3 and 4 show the results of washed versus unwashed monolayers,respectively. These data demonstrate that washing of cell monolayersfour hours after infection did not alter outcome and that thiostreptonappears to be consistently effective in all assays compared with PRM. Aconsistent cytotoxicity of infected medium and medium with oocyst lysateis also indicated.

Experiment 3

In vitro doses of drugs. Thiostrepton (Calbiochem), Phavic (NTZ) weredissolved in sterile complete Dulbecco's Modified Eagle Medium (DMEM)supplemented with dimethyl sulfoxide (DMSO) at 0.2%. Thiostrepton wastested at a concentration of 800 μg/ml. NTZ was tested at aconcentration of 10 μg/ml. Paromomycin was dissolved in DMEM and testedat 2000 μg/ml. Cell monolayers were infected and washed, followed by theaddition of drug at time intervals of 0, 2, 4, 8 and 24 hours.

Toxicity Testing Assay. 200 μl of medium containing drug at theabove-mentioned concentrations and positive control preparations[paromomycin/DMSO (2 mg/ml/0.2%)] were introduced into two wells of a 96well plate containing confluent MDBKF5D2 cell monolayers infected withintact C. parvum GCH1 oocysts (5.0×10⁴ per well) and two wells withoutmonolayers. The drug was incubated on the monolayers at 37° C. and 8%CO₂. At 48 hours, MTS (Owen's solution) and PMS were added to each wellat concentrations of 333 μg/ml and 25μM respectively. The plate wasreturned to the incubator in the dark to develop for two hours. At twohours, 100 μl of each supernatant was transferred to a new microtiterplate and the optical density (OD) was read in an ELISA plate reader at490 nm.

Percent toxicity was calculated by subtracting the mean drug OD from themedium OD, divided by the medium OD, all of which was then multiplied by100, as shown in the equation below. Cytotoxicity scores were assignedas follows: 0-5% toxicity=0, 6-25% toxicity=1, 26-50% toxicity=2, 51-75%toxicity=3, and 76-100% toxicity=4. As a standard, cytotoxicity scoresof 0 or 1 were considered acceptable levels of toxicity. Toxicity scoresof 2, 3, or 4 were considered as high levels of toxicity to the cellmonolayer.$\frac{{O\quad D\quad {medium}} - {O\quad D\quad {drug}}}{O\quad D\quad {medium}} \times 100$

Intact C. parvum oocyst assay. 3.0×10⁴ C. parvum GCH1 oocysts per wellwere incubated in the above-mentioned concentrations of drug at 37° C.(8% CO₂) on confluent MDBKF5D2 cell monolayers in 96 well microtiterplates. The level of infection in each well was determined and analyzeby immunofluorescence assay at 48 hours, using C. parvum sporozoiterabbit anti-serum (0.1%) and fluorescein-conjugated goat anti-rabbitantibody (1.0%). Percent inhibition was calculated by subtracting themean parasite/drug from the mean parasite/medium, divided by the meanparasite/medium, all of which was multiplied by 100. The analysis wasperformed using MCID and an inverted microscope.

Data presented in Tables 5 and 6 demonstrate that PRM is well within thenormal range and thiostrepton appears to be consistently effective.Washing of monolayers four hours after infection did not alter theoutcome. Consistent cytotoxicity levels of the infected medium andmedium with oocyst lysate were also noted. These data show that boththiostrepton and NTZ act on the intracellular forms of the parasite, inmuch the same way as does PRM. However, unlike PRM, whose activity dropswhen it is added 24 after infection, both thiostrepton and NTZ werestill highly inhibitory even i24 hours after infection.

In summary, thiostrepton was compared to paromomycin (PRM; usedclinically for treatment) and Phavic (NTZ), also under development foractivity against Cryptospordium parvum. For in vitro testing, thecompounds were added at the time of cell invasion, so both sporozoiteand intracellular stages were exposed. The drugs were also tested attime points following invasion, when only intracellular stages arepresent. Briefly, unlike PRK, both thiostrepton and NTZ were highlyinhibitory even 24 hours after infection. This is consistent withthiostrepton targeting the plastid-like organelle as there should be nostage -specificity. Thiostrepton has also been shown to be moreeffective than PRM at about one-fifth the dose, with little toxicity tocells.

In vivo Testing

SCID mice (Taconic Farms, Germantown, N.Y.), preconditioned withmonoclonal antibodies to interferon as described (41), were given anacute infection of C. parvum (10⁷oocysts given orally). A total of fivemice in two groups were used. One group received 500 mg/kg/day (in twodoses of 250 mg/kg) of thiostrepton six days post-infection and theother group received a placebo. Treatment lasted ten days and oocystshedding was measured by counting the number of oocysts in 30 fieldsunder high power microscopy of acid-fast stained fecal smears andcalculating the mean for each group as described (41). The results inFIG. 1 showed that thiostrepton significantly reduced oocyst shedding(shaded bars) compared to placebo (black bars). These data aresummarized in Table 7. Mucosal scores describe the extent of mucosalinfection detected in formalin-fixed sections of tissue samples fromvarious gut sites (pyloric region of stomach, liver, gallbladder,mid-small intestine, terminal ileum, cecum and colon) taken duringnecropsy. Scoring was as follows: 0 (no infection) to 5 (maximalinfection) and was expressed as the combined score of the number of gutsites examined.

III. Methods for Treating Parasitic Infections in Humans and Non-humanAnimals

Treatment of parasitic infection in humans and non-human animals. Totreat a parasitic infection in a human or non-human animal diagnosed ashaving an infection by a parasite having a plastid-like organelle,approximately 500 mg/kg/day of thiostrepton can be administered as asingle dose or in multiple doses to the infected individual, eitherorally or parenteraly in a pharmaceutically acceptable carrier. Thedaily administration can be continued for an indefinite period for aslong as signs and symptoms of the parasitic infection persist. The signsand symptoms of the infected individual which can be monitored includeamount and frequency of diarrhea excretion, quantitation of oocystexcretion, body weight, overall appearance and condition, bloodchemistry and urine analysis of the infected individual's hepatobiliaryfunction and culture of the parasite from body fluids and tissues.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

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TABLE 1 Conc. Parasite Standard Tox. Standard Inhibition Toxicity Drug(μg/ml) Count Deviation O.D. Deviation Percent Score Percent ScoreMedia/DMSO 1037.56 176.02 1.100 .346 00 0 00 0 PRM/DMSO 2000 μg/ml278.06 47.30 .868 .071 73.20 3 21.09 1 Media-Infected NA NA .928 .199 NANA 15.64 1 Media-Lysed NA NA .852 .051 NA NA 22.55 1 Thiostrepton 100μg/ml 732.31 54.80 .994 .161 29.42 0 9.64 1 10 909.25 128.71 .991 .13412.37 0 9.95 1 1 1193.19 57.34 .941 .350 <0 0 14.50 1 .1 1052.13 104.48.912 .029 <0 0 17.14 1 Conc. - units = μg/ml; Parasite - Mean parasitecount/field (16 fields at 10x mag. analyzed) SD - Standard Deviation; %Inhib. - Percent inhibition of parasite infection % Tox - Percenttoxicity to cells by the drug; *NA - Not available due to toxicityScores - expressed as range from 1 (non-toxic) to 4 (very toxic).Percent inhibition scores - expressed in ranges as 0: 0-30%; 1: 31-55%;2: 56-70%; 3: 71-90%; and 4: 91-100%

TABLE 2 Conc. Parasite Standard Tox. Standard Inhibition Toxicity Drug(μg/ml) Count Deviation O.D. Deviation Percent Score Percent ScoreMedia/DMSO 1220.25 197.59 1.007 .008 .00 0 .00 0 PRM/DMSO 2 mg/ml 369.81101.90 .963 .157 69.69 2 4.37 0 Thiostrepton 800 μg/ml 149.25 27.49 .676.049 87.77 3 32.87 2 400 334.06 86.98 .799 .006 72.62 3 20.71 1 200587.81 140.82 .820 .185 51.83 1 18.62 1 100 943.31 99.28 .915 .143 22.700 9.14 1 Media 1811.69 376.93 .914 .010 .00 0 .00 0 PRM 2 mg/ml 423.81100.96 .954 .012 76.61 3 −4.38 0

TABLE 3 Unwashed Conc. Parasite Standard Tox. Standard InhibitionToxicity Drug (μg/ml) Count Deviation O.D. Deviation Percent ScorePercent Score Media 1416.44 301.91 1.043 .303 .00 0 .00 0 Media/DMSO1231.75 280.96 1.031 .116 .00 0 1.06 0 Thiostrepton 800 μg/ml 106.0645.04 .739 .231 91.39 4 29.11 2 400 154.88 32.94 .788 .055 87.43 3 24.411 200 391.19 107.54 .827 .122 68.24 2 20.67 1 100 795.63 166.53 .969.122 35.41 1 7.10 1 PRM 2000 256.38 64.76 1.061 .290 81.90 3 −1.73 01000 293.56 96.74 .963 .097 79.27 3 7.67 1 500 398.31 87.14 1.019 .05971.88 3 2.30 0 250 453.94 74.96 1.121 .165 67.95 2 −7.53 0 NTZ 100 NA NA.269 .057 NA NA 74.15 3 10 87.31 20.74 1.302 .338 92.91 4 −24.94 0 1694.75 172.52 .925 .129 43.60 1 11.32 1 .1 1104.81 127.09 .855 .02710.31 0 17.99 1 Media-Inf NA NA .836 .005 NA NA 19.81 1 Media-Lysate NANA .859 .040 NA NA 17.55 1

TABLE 4 Washed at 4 hours Conc. Parasite Standard Tox. StandardInhibition Toxicity Drug (μg/ml) Count Deviation O.D. Deviation PercentScore Percent Score Media 1210.28 209.00 1.041 .116 .00 0 .00 0Media/DMSO 1395.69 184.63 1.033 .303 .00 0 .77 0 Thiostrepton 800 176.3125.36 .739 .231 87.37 3 29.01 2 400 164.31 28.04 .788 .055 88.23 3 24.301 200 297.19 52.99 .827 .122 78.71 3 20.56 1 100 543.88 100.53 .969 .12261.03 2 6.96 1 PRM 2000 211.38 70.20 1.061 .290 82.54 3 −1.87 0 1000251.88 83.69 .963 .097 79.19 3 7.54 1 500 372.44 89.66 1.019 .059 69.232 2.16 0 250 492.13 226.63 1.121 .165 59.34 2 −7.68 0 NTZ 100 NA NA .269.057 NA NA 74.11 3 10 96.06 35.55 1.302 .338 93.12 4 −25.12 0 1 233.4452.92 .925 .129 83.27 3 11.19 1 .1 1334.06 318.47 .855 .027 4.42 0 17.871 Media-Inf NA NA .836 .005 NA NA 19.69 1 Media-Lysate NA NA .859 .040NA NA 17.44 1

TABLE 5 Conc. Parasite Standard Tox. Standard Inhibition Toxicity Drug(μg/ml) Count Deviation O.D. Deviation Percent Score Percent Score Media1273.06 209.80 1.148 .078 .00 0 .00 0 Media/DMSO 960.19 178.72 1.015.129 .00 0 .00 0 PRM 0 hrs 2000 μg/ml 287.63 30.61 .962 .137 77.41 316.17 1 2 hrs 2000 134.94 32.98 .878 .058 89.40 3 23.49 1 4 hrs 2000267.25 46.78 .923 .123 79.01 3 19.56 1 8 hrs 2000 354.06 71.53 .871 .17672.19 3 24.05 1 24 hrs 2000 929.19 123.61 .850 .175 27.01 0 25.93 1Thiostrepton 0 hrs 800 112.44 23.10 .783 .002 91.17 4 22.91 1 2 hrs 800104.19 30.87 .577 .136 91.82 4 43.15 2 4 hrs 800 196.81 73.44 .638 .08584.54 3 37.14 2 8 hrs 800 192.56 35.92 .786 .048 84.87 3 22.56 1 24 hrs800 318.25 42.22 .749 .138 75.00 3 26.26 2 NTZ 0 hrs 10 113.06 24.441.236 .120 91.12 4 −21.72 0 2 hrs 10 53.94 13.71 .900 .032 95.76 4 11.381 4 hrs 10 112.31 37.04 .952 .108 91.18 4 6.16 1 8 hrs 10 178.13 42.781.014 .008 86.01 3 .10 0 24 hrs 10 501.63 139.70 .990 .005 60.60 2 2.410 Media-Inf NA NA .997 .110 NA NA 13.15 1 Media-Lysate NA NA 1.086 .043NA NA 5.44 0

TABLE 6 Conc. Parasite Standard Tox. Standard Inhibition Toxicity Drug(μg/ml) Count Deviation O.D. Deviation Percent Score Percent ScoreMedia/DMSO 1372.19 16.00 1.647 .001 .00 0 .00 0 PRM/DMSO 2 mg/ml 325.7587.10 1.273 .112 76.26 3 22.74 1 Thiostrepton 800 μg/ml 223.13 37.38.890 .035 83.74 3 45.96 2 400 288.13 53.38 1.146 .124 79.00 3 30.45 2200 470.69 151.68 1.324 .185 65.70 2 19.61 1 100 871.25 139.37 1.348.242 36.51 1 18.15 1

TABLE 7 Oocyst shedding-last day of 10 day treatment Groups of 5 Mean+SD % Inhib Mucosal score +SD Group 1 (placebo) 53.2 15.8 — 19.3 7.6Group 2 (2 × 250 11.2 5.1 78.9 9.8 5.3 mg/kg)

8 19 base pairs nucleic acid single linear not provided 1 GGGCTTTAGAAGCTTTTGG 19 22 base pairs nucleic acid single linear not provided 2CCATTTAAAA TTGGTAATCC TG 22 18 base pairs nucleic acid single linear notprovided 3 GTGTGATAAT ATTCATGG 18 15 base pairs nucleic acid singlelinear not provided 4 GGAGAGCATT TGGTG 15 22 base pairs nucleic acidsingle linear not provided 5 GTTTAGCTAT TAATATAGAA GC 22 18 base pairsnucleic acid single linear not provided 6 CGGAGAGGTA TTAATACC 18 20 basepairs nucleic acid single linear cDNA not provided 7 AAACTTGTGTTCGGATATAG 20 18 amino acids amino acid single linear peptide notprovided 8 Ile Ala Ser Ala Ser Cys Thr Thr Cys Ile Cys Thr Cys Ser Cys 15 10 15 Ser Ser Ser

What is claimed is:
 1. A method for treating a parasitic infection in asubject infected with a parasite having a plastid-like organelle,comprising administering to the subject an infection treating amount ofa thiopeptide in a pharmaceutically acceptable carrier, wherein thesubject is a mammal.
 2. The method of claim 1, wherein the mammal is ahuman.
 3. The method of claim 1, wherein the parasite is from theApicomplexa group of parasites.
 4. The method of claim 3, wherein theparasite is selected from the group consisting of Plasmodium, Toxoplasmaand Cryposporidium species.
 5. The method of claim 4, wherein theparasite is Cryptospordium.
 6. The method of claim 4, wherein theparasite is Toxoplasma.
 7. The method of claim 4, wherein the parasiteis Plasmodium.
 8. The method of claim 1, wherein the thiopeptide isselected from the group consisting of thiostrepton, micrococcin,nosiheptide, siomycin, sporangiomycin, althiomycin, thiocilin andthiopeptin.
 9. The method of claim 8, wherein the thiopeptide isthiostrepton.
 10. The method of claim 1, wherein the thiopeptide isadministered to the subject orally.
 11. The method of claim 1, whereinthe thiopeptide is administered to the subject parenterally.
 12. Amethod for treating Cryptoporidium infection in a subject infected witha parasite having a plastid-like organelle comprising administering tothe subject an infection treating amount of a thiopeptide in apharmaceutically acceptable carrier, wherein the subject is a mammal.13. The method of claim 12, wherein the mammal is a human.
 14. Themethod of claim 12, wherein the thiopeptide is selected from the groupconsisting of thiostrepton, micrococcin, nosiheptide, siomycin,sporangiomycin, althiomycin, thiocillin and thiopeptin.
 15. The methodof claim 14, wherein the thiopeptide is thiostrepton.
 16. The method ofclaim 12, wherein the thiopeptide is administered orally.
 17. A methodfor treating a parasitic infection in a subject infected with a parasitehaving a plastid-like organelle, comprising administering to the subjectan infection treating amount of a thiopeptide in a pharmaceuticallyacceptable carrier, wherein the parasitic infection is caused by amember of the Microspora phylum or Ascetospora phylum.
 18. The method ofclaim 17, wherein the subject is a mammal.
 19. The method of claim 18,wherein the mammal is a human.
 20. The method of claim 17, wherein thethiopeptide is selected from the group consisting of thiostrepton,micrococcin, nosiheptide, siomycin, sporangiomycin, althiomycin,thiocillin, and thiopeptin.
 21. A method for treating a subject infectedwith a parasite comprising administering a thiopeptide to the subject,wherein the parasite is selected from the group consisting ofPlasmodium, Toxoplasma, and Cryptospordium.
 22. The method of claim 21wherein the parasite is Plasmodium.
 23. The method of claim 21 whereinthe parasite is Toxoplasma.
 24. The method of claim 21 wherein theparasite is Cryptospordium.